WO2015001963A1

WO2015001963A1 - Separation device and separation method

Info

Publication number: WO2015001963A1
Application number: PCT/JP2014/066232
Authority: WO
Inventors: 啓介渋谷; 靖彦多田; 近藤　健之; 聖村上
Original assignee: 株式会社日立製作所
Priority date: 2013-07-05
Filing date: 2014-06-19
Publication date: 2015-01-08
Also published as: US20160136543A1; EP3018475B1; US10086313B2; JP6203557B2; EP3018475A4; EP3018475A1; JP2015014539A

Abstract

[Problem] To separate a material to be separated at a low cost and constant accuracy when the material to be separated is separated from a mobile phase containing the material to be separated through the passing of the mobile phase through a stationary phase, even if the mobile phase has a large volume. [Solution] A separation device characterized in that a separation column provided with a stationary phase having a volume capable of processing the entire volume of a mobile phase containing a material to be separated is provided, the separation column is replaceable, and the usage count of the stationary phase reaches a lifetime count through the processing of one batch.

Description

Separation apparatus and separation method

The present invention relates to a separation apparatus and a separation method for separating a substance from a mobile phase by passing a mobile phase containing the substance to be separated through a stationary phase.

The separation device that separates the substance from the mobile phase by allowing the mobile phase containing the substance to be separated to pass through the stationary phase is used, for example, when separating useful substances produced by cell culture. For example, an antibody drug which is a useful substance can be obtained by culturing animal cells having antibody-producing ability, and separating and purifying the antibody secreted in the culture solution. That is, useful substances such as antibody drugs are separated and purified using chromatography after removing cells from the culture solution. On the other hand, when a predetermined useful substance is produced using microorganisms, the useful substance is often accumulated in cells. In this case, useful substances are separated and purified using chromatography after removing solids from the solution after disrupting the cells.

Generally, antibody drugs are produced through a clarification process, a recovery process, an intermediate purification process, and a final purification process. In this production method, different separation apparatuses, that is, chromatographic techniques are used in each step depending on the type of antibody of interest. However, both are common in that the purity of the antibody is increased stepwise and the selectivity of the target antibody is increased.

In the clarification step, proteins and solids other than antibodies are removed from the culture solution as much as possible. Culturing is performed using salting out, filtration through a filter, centrifugation, etc., because the components of the culture fluid such as serum, ascites fluid, and hybridoma cell culture fluid are different for each type of antibody, and the contents are also different.

In the recovery step, affinity chromatography is usually used. When the target antibody is IgG, affinity chromatography with very high specificity using Protein A or Protein G as a ligand is used, and purification with a purity of 90% or more is possible in one step. On the other hand, for the purpose of antibodies having low affinity with Protein A or G, such as IgM or IgY, affinity chromatography using Thiophilic interaction is used. For purposes of IgA, IgD, and IgE, affinity chromatography with a secondary antibody that recognizes the antibody is used because of its low blood concentration and lack of high affinity affinity ligands. . In this recovery step, processing speed and processing volume are important, and it is required to quickly separate and concentrate the target antibody from a crude state such as a cell extract. This is to facilitate subsequent steps.

Next, in the intermediate purification process, impurities collected together with the target antibody in the collection process are removed. In this step, since the amount of the treatment solution is large, ion exchange chromatography having a large treatment amount is generally used. When the amount of the processing solution is small, the recovery step and the intermediate purification step can be performed in one step.

The final final purification step is a step for separating slightly remaining impurities using a high performance column to obtain a final purified antibody. In the final purification step aimed at antibodies, gel filtration chromatography using a column with high resolution is generally used. By using gel filtration chromatography, it is possible to exchange the buffer together with the removal of the low molecular weight substance that inhibits the structural analysis.

In the production of biopharmaceuticals, generally, a plurality of culture tanks having a capacity of about 10 m ³ are installed, and after culturing animal cells in these culture tanks, a large amount of culture solution containing biopharmaceuticals (eg, antibodies) (1 batch) 10m ³ or more). A particular problem with such a large amount of culture solution treatment is a recovery step using affinity chromatography. Proteins that specifically bind to biopharmaceuticals (such as Protein A) used in affinity chromatography are very expensive. Therefore, the column using the protein is repeatedly reused for each culture batch. However, when the column is reused, various components in the previous process may remain, and the column may be deteriorated. Due to these, the purification quality of biopharmaceuticals may differ between culture batches.

In general, a purified affinity column for producing biopharmaceuticals is very large with a diameter of about 1 m and a bed height of about several tens of centimeters. On the other hand, since the culture solution to be processed is as large as 10 m ³ or more, even if the large purification column is used, it is necessary to repeat the collection step many times in order to process one batch of culture solution. The recovery process includes (I) equilibration of the column, (II) adsorption of the target substance, (III) washing, (IV) elution of the target substance, and (V) regeneration of the column. The step of performing (II) is the step of adsorption of the target substance, and the steps (I) and (III) to (V) are in a waiting state for treating the culture solution. In order to process a large amount of culture solution, the recovery steps (I) to (V) above must be repeated many times, resulting in an increase in waiting time, resulting in an increase in processing time in the recovery step. . For example, in Patent Document 1, in order to eliminate waiting time, a plurality of columns are installed, and the recovery process in each column is shifted to treat the culture solution. (II) The target substance adsorption process is continuously performed. Methods of processing are being considered. However, this method has a problem that it is necessary to secure a wider facility space because a plurality of columns are installed, and the cost of the column packing is increased.

JP 2011-214837 A

As described above, conventionally, when a mobile phase containing a substance to be separated is passed through a stationary phase, the substance is separated from the mobile phase at a low cost and with a certain accuracy even for a large volume mobile phase. It is an object of the present invention to provide a separation apparatus and a separation method capable of separating a substance to be separated.

As a result of intensive studies by the present inventors in order to achieve the above-mentioned object, by providing a stationary phase having a capacity necessary for processing a predetermined capacity of the mobile phase, it is possible to change the stationary phase every batch. It has been found that the target substance can be separated with high accuracy and the processing time for processing a plurality of batches can be shortened, and the present invention has been completed.

That is, the present invention includes the following.
(1) A separation column having a stationary phase with a capacity capable of treating the entire amount of the mobile phase containing the substance to be separated is provided, the separation column is replaceable, and the number of times the stationary phase is used in one batch processing. Separation device characterized by the number of lifetimes.
(2) The separation column includes a plurality of columns including the stationary phase, and the total amount of the stationary phase packed in the plurality of columns is a capacity capable of processing the total amount of the mobile phase containing the substance to be separated. (1) The separation device according to (1).
(3) A plurality of pipes connected to each of the plurality of columns, a plurality of switch valves respectively disposed on the plurality of pipes, and communication of each pipe by the plurality of switch valves are controlled. The separation device according to (2), further comprising a control device.
(4) The separation apparatus according to (2), wherein the total column bed height of the plurality of columns is a column bed height when the stationary phase having the capacity is a single column.
(5) The separation apparatus according to (1), wherein the capacity of the stationary phase is defined based on the total amount of substances to be separated contained in the mobile phase and the maximum adsorption capacity of the stationary phase.
(6) A separation column having a stationary phase with a capacity capable of treating the entire mobile phase containing the substance to be separated, the separation column being exchangeable, and the number of times the stationary phase is used in one batch of treatment. Supplying the mobile phase to the separation column having a lifetime of
Recovering the substance to be separated from the stationary phase after treating the total amount of the mobile phase,
A separation method characterized by exchanging a used separation column after the recovery step.
(7) The separation column includes a plurality of columns including the stationary phase, and the total amount of the stationary phase packed in the plurality of columns is a capacity capable of processing the total amount of the mobile phase containing the substance to be separated. Yes,
The separation method according to (6), wherein the mobile phase is sequentially supplied to the plurality of columns.
(8) A plurality of pipes are connected to each of the plurality of columns, a plurality of switch valves are arranged on the plurality of pipes, and a control device controls communication of each pipe by the plurality of switch valves. (6) The separation method according to (7).
(9) The separation method according to (7), wherein the sum of the column bed heights of the plurality of columns is a column bed height when the stationary phase having the capacity is a single column.
(10) The separation method according to (6), wherein the capacity of the stationary phase is defined based on the total amount of substances to be separated contained in the mobile phase and the maximum adsorption capacity of the stationary phase.
(11) a tank storing a mobile phase containing a substance to be separated;
The separation column is connected to the tank and includes a stationary column having a volume capable of treating the entire amount of the mobile phase stored in the tank, and the separation column is replaceable. An apparatus for producing a substance, characterized in that the number of times of use becomes the number of lifetimes.
(12) The separation column includes a plurality of columns including the stationary phase, and the total amount of the stationary phase packed in the plurality of columns is a capacity capable of processing the total amount of the mobile phase including the substance to be separated. (11) The substance production apparatus according to (11).
(13) A plurality of pipes connected to each of the plurality of columns, a plurality of switch valves respectively disposed on the plurality of pipes, and communication of each pipe by the plurality of switch valves are controlled. The apparatus for producing a substance according to (12), further comprising a control device.
(14) The apparatus for producing a substance according to (12), wherein the sum of the column bed heights of the plurality of columns is a column bed height when the stationary phase having the capacity is a single column. (15) The capacity of the stationary phase is defined based on the total amount of substances to be separated contained in the mobile phase and the maximum adsorption capacity of the stationary phase, The substance production apparatus according to (11) .

According to the separation apparatus and the separation method of the present invention, the target substance can be separated with a certain accuracy for each mobile phase batch to be processed. Therefore, by using the separation apparatus according to the present invention, a high-quality target substance with little variation in quality can be obtained.

It is a schematic block diagram which shows an example of the separation apparatus to which this invention is applied. It is an example of the block diagram of a continuous affinity refinement | purification apparatus (3 columns). It is another example of the block diagram of a continuous affinity refinement | purification apparatus (3 columns). It is an example of the block diagram of a continuous affinity refinement | purification apparatus (2 columns). It is another example of the block diagram of a continuous affinity refinement | purification apparatus (3 columns). It is a figure which shows the example of the process of a continuous affinity refinement | purification apparatus. It is a figure which shows the relationship between column bed height and a linear velocity. It is a figure which shows how to obtain | require dynamic coupling capacity. It is a figure which shows the example of a division | segmentation of a column. It is a figure which shows the refinement | purification system applied to the biopharmaceutical manufacturing plant.

Hereinafter, the present invention will be described in detail.
As shown in FIG. 1, the separation apparatus to which the present invention is applied comprises a separation column C filled with a stationary phase. When the solution B (mobile phase) stored in the tank A is supplied to the separation column C, the substance to be separated contained in the solution is captured by the stationary phase in the separation column C. Thereafter, when the eluent is supplied to the separation column C, the substance trapped in the stationary phase is recovered from the separation column C. The stationary phase packed in the separation column C has a capacity capable of processing the entire amount of the solution to be processed (mobile phase), that is, a capacity that reaches the number of lifetimes by processing for one batch. Therefore, by replacing the separation column C for each batch of the solution B to be treated, the separation apparatus to which the present invention is applied, the processing time when processing a plurality of batches can be shortened. Variations in separation accuracy caused by stationary phase degradation and the like can be suppressed. Since the separation column and the separation control device portion are separated by the column connection joint portion, the separation column C can be easily replaced by simply removing the column connection joint.

Here, the treatment for one batch means, for example, a predetermined amount of solution obtained by one batch culture when the target substance is produced by cell culture. However, the treatment for one batch is not limited to the total amount of the solution obtained by one batch culture, and may be a half amount of the solution obtained by one batch culture, or one fed-batch culture. Or a half amount of the solution obtained by one fed-batch culture.

Here, the number of lifetimes of the stationary phase means the number of times that the recovery amount of the target substance by the stationary phase is reduced to a predetermined ratio. For example, the recovery amount of the target substance is 10%, 20%, 30%, 40 %, 50%, 60%, 70%, 80%, 90% or 95%. In particular, the number of times the recovery amount of the target substance is reduced to 95% is preferably the life cycle number, and the number of times the recovery amount of the target substance is reduced to 90% is more preferably the life cycle number. It is most preferable that the number of times the life is reduced to 80% be the life number.

Hereinafter, an example of a separation apparatus to which the present invention is applied will be described more specifically. As described above, the separation device refers to the specific substance from other substances in the mobile phase by passing the mobile phase through the stationary phase and capturing the specific substance in the mobile phase in the stationary phase. Means a device to separate. The separation device may be referred to as a purification device, and is synonymous with a so-called chromatography device. The separation device may be any of so-called partition chromatography, adsorption chromatography, molecular exclusion chromatography, ion exchange chromatography, and affinity chromatography. In the following example, a system for separating a protein containing an antibody used in medicine or the like from a cell culture medium will be described. However, the separation apparatus according to the present invention is not limited to this example, and any substance may be used as the substance to be separated.

<Separator>
A preferred embodiment of the separation apparatus according to the present invention is shown in FIGS. The separation apparatus shown in FIGS. 2 and 3 includes three affinity packed

columns

1A, 1B, and 1C (

separate columns

1A, 1B, and 1C) that continuously process a solution containing a substance to be separated (mobile phase, sample solution). And various solutions used in the separation process, a pump for feeding the liquid, and the like. In the separation apparatus, the number of packed columns is not limited to three, and there may be two or more packed columns as required as shown in FIG. Here, the apparatus configuration in the case of three packed columns (FIGS. 2 and 3) will be described in detail.

The separation apparatus shown in FIG. 2 includes three

separation columns

1A, 1B and 1C connected in series, five liquid-

feeding pumps

3 and 4, five

solution reservoirs

5, 6, 7, 8 and 9, and a switch. It is comprised from the valve | bulb 15 (flow-path switching site | part apparatus), and the piping which connects them. The columns connected in series have a structure in which the upstream and downstream are connected by piping and can be circulated. Upstream of each column can be passed through various solution reservoirs and a switch valve 15. Further, downstream of each column is a structure in which liquid can be passed through the switch valves 41 to 43 to the switch valve 38, and the switch valve 38 is connected to the waste liquid pipe or the purified solution pipe.

The various solutions that have entered the

reservoirs

5, 6, 7, 8, and 9 are fed to the switch valve 15 by driving the liquid feeding pumps 3 and 4. The switch valve 15 switches the flow path so that various solutions are sent to

appropriate separation columns

1A, 1B, and 1C according to the process shown in FIG. In order to process all the

separation columns

1A, 1B and 1C simultaneously, the switch valve 15 is configured so that all the one-to-one combinations of various solutions and flow path connections to the

separation columns

1A, 1B and 1C can be performed. Yes. However, when there is a solution that is not sent as a process to each column, there is also a solution that does not feed (does not operate the feed pump 3 or 4).

In the case of the target substance separated by the eluate, the solution coming out from each of the

separation columns

1A, 1B, and 1C is recovered as a purified solution through a recovery pipe, and otherwise is recovered as a waste liquid from the waste liquid pipe. . Each

separation column

1A, 1B and 1C and each pipe (purification and waste liquid pipe) are selected by the switch valve 38 according to the process shown in FIG. The

switch valves

15 and 38 are configured so that each

separation column

1A, 1B and 1C and each pipe realize all combinations of flow paths. In order to control the

pumps

3 and 4, the

switch valves

15 and 38, etc. according to the steps shown in FIG. 6, a control program can be stored in the computer 39 in advance and executed by the control device 40 in accordance with the control program. Thereby, by using this separation apparatus, a sample solution can be processed continuously and only a target substance can be collected continuously. In order to confirm whether the target substance is properly recovered, it can also be detected by absorbance with a UV device.

This device can perform a simulated moving bed process, and the specific steps are described below. Step 1: A feed solution (reservoir 5) containing at least one target compound is passed across the separation column 1A and the effluent is moved from the separation column 1A to the separation column 1B. Step 2: The switch valve 15 is controlled so that the feed solution (reservoir 5) is sent to the separation column 1B, and the washing solution (reservoir 6) is passed across the separation column 1A to which the target compound is bound. Step 3: The washing liquid effluent (downstream of the separation column 1A) is moved to the separation column 1C, and then the effluent from the separation column 1B is moved to the separation column 1C. Step 4: Regenerate the separation column 1A. Step 5: Direct feed solution (reservoir 5) to separation column 1C and pass wash solution (reservoir 6) across separation column 1B to which the target compound is bound. Step 6: The washing liquid effluent (downstream of the separation column 1B) is moved to the separation column 1A, and then the effluent from the separation column 1C is moved to the separation column 1A. Step 7: Regenerate the separation column 1B. Step 8: Direct feed solution (reservoir 6) to separation column 1A and pass wash solution (reservoir 6) across separation column 1C to which the target compound is bound. Step 9: The washing liquid effluent (downstream of the separation column 1C) is moved to the separation column 1B, and then the effluent from the separation column 1A is moved to the separation column 1B. Step 10: Regenerate the separation column 1C. Then, steps 2 to 10 are repeated according to the steps shown in FIG. Here, at least one substance to be separated is recovered in Step 4, Step 7, and / or Step 10.

On the other hand, a separation apparatus as shown in FIG. 3 may be used. Three

separation columns

1A, 1B and 1C connected in parallel (a column connected in series is regarded as one column), five liquid-

feeding pumps

3 and 4, five

solution reservoirs

5, 6, 7, 8 and 9, a switch valve 15 (flow path switching part device), and a pipe connecting them. The various solutions that have entered the

reservoirs

appropriate separation columns

separation columns

separation column

switch valves

15 and 38 are configured so that each

separation column

pumps

3 and 4, the

switch valves

FIG. 4 shows a separation apparatus having two

separation columns

1A and 1B, and FIG. 5 shows a separation apparatus having three

separation columns

1A, 1B and 1C, and three liquid feed pumps 3, A separation device having 4 and 34 is illustrated.

4 includes two separation columns (column 1A and column 1B), two liquid-feeding pumps (pump 3 and pump 4), solution reservoirs (reservoir 5, reservoir 6, reservoir 7, and reservoir 8). , Reservoir 9), flow path switching site device (switch valve 10, switch valve 11, switch valve 12, switch valve 13, selection valve 14, selection valve 15), and piping connecting them.

The solution stored in the reservoir 5 is fed by the pump 3 and can be fed to the piping of the switch valve 21 or the piping of the switch valve 22 by the control of the selection valve 15. The solution stored in the reservoir 6, the reservoir 7, the reservoir 8, and the reservoir 9 can be fed by the pump 4, and the solution fed to the pipe connected to the pump 4 can be selected by the control of the selection valve 14. it can. By controlling the selection valve 15, it is possible to control whether the solution sent to the pump 4 is sent to the pipe on the switch valve 23 side or the pipe on the switch valve 24 side. In the selection of the selection valve 15, when the piping from the pump 3 side is selected to the switch valve 21 side, the piping on the pump 4 side is simultaneously selected to the switch valve 24 side, or the piping from the pump 3 side is When selected on the switch valve 22 side, the piping on the pump 4 side is simultaneously selected on the switch valve 23 side.

In the switch valve 10, either the column 1A side pipe or the waste liquid side pipe is selected, and in the switch valve 11, either the column 1B side pipe or the waste liquid side pipe is selected. The column 1A is connected to the switch valve 12 through a pipe on the opposite side to the switch valve 10. The column 1B is connected to the switch valve 13 via a pipe on the opposite side of the switch valve 11. In the

switch valves

12 and 13, either the pipe on the collection container 25 side or the pipe on the waste liquid side is selected.

In the above-described separation device, all pumps and flow path switching devices (switch valves) are controlled by a control device in which liquid feeding conditions such as liquid feeding timing, liquid feeding speed and liquid feeding time are programmed in advance. That is, the separation operation can be performed automatically and continuously by operating the pump and the flow path switching device in a predetermined order by the control device. In addition, although the number of reservoirs connected to the pump 4 by the selection valve 14 is four in the example of FIG. 4, it may be more or less.

In FIG. 4, switch

valves

10 and 11 arranged immediately upstream of the

columns

1A and 1B flow the solution flowing through the

columns

1A and 1B to the waste liquid side in advance, respectively, and switch the solutions leading to the

columns

1A and 1B. In this case, the mixing of the solution before and after can be minimized. The

switch valves

12 and 13 disposed immediately downstream of the

columns

1A and 1B allow the solution coming out of the

columns

1A and 1B to pass through the waste reservoir container or the recovery container 25, respectively. Can be switched. Reservoir 5, reservoir 6, reservoir 7, reservoir 8, and reservoir 9 are various solution reservoirs. The reservoir 5 contains a solution (mobile phase) containing a substance to be separated, and a selection valve 15 via a liquid feed pump 3. To be supplied. The reservoir 6 contains a column equilibration solution, the reservoir 7 contains a washing solution, the reservoir 8 contains an elution solution, and the reservoir 9 contains a regeneration solution. The selection valve 14 can switch any one of the reservoir 6, the reservoir 7, the reservoir 8, and the reservoir 9 to communicate with each other, and can supply various solutions to the selection valve 15 via the liquid feeding pump 4.

The selection valve 15 is a valve for switching the solution flowing from the pump 3 side and the solution flowing from the pump 4 side to the column 1A or the column 1B while selecting the flow path so as not to select the same column at the same time. is there. For example, when the flow from the pump 3 side flows to the column 1A side, the switch valves 21 to 24 are controlled so that the flow from the pump 4 side flows to the column 1B side. Further, when the flow from the pump 3 side flows to the column 1B side, the switch valves 21 to 24 are controlled so that the flow from the pump 4 side flows to the column 1A side. The purified protein separated by the

column

1A or 1B can be intermittently recovered by sending it to the recovery container 25 only when it is desired to recover it by the switch valve 12 and the switch valve 13. In addition, in order to monitor whether collection | recovery is performed normally, chromatography monitoring can be performed by attaching the ultraviolet monitor 26 (UV monitor) in the middle of a collection line.

Although the case where four types of reservoir 6, reservoir 7, reservoir 8, and reservoir 9 are used is shown in the figure, it is possible to perform chromatography using four or more types of solutions. In this example, as the solution required in the adsorption chromatography, an equilibration solution, a washing solution, an elution solution, and a regeneration solution are required, and therefore, the minimum unit configuration is described. When four or more types of solutions are used, a reservoir and a switch valve corresponding to the number of solutions to be used may be used in accordance with the configuration shown in FIG. Even in this case, on / off control of the pump, switching of the flow rate and valve, etc. can be controlled in advance by a control device such as a computer. Therefore, by using this apparatus, after starting the liquid feeding of the sample, it is continuously operated until the sample runs out, and the protein can be purified by full automation.

Further, in the separation device shown in FIG. 5, unlike the separation device shown in FIG. 4, the solution filled in the

reservoirs

6 and 7 is sent through the selection valve 14 and the pump 4. Further, the separation apparatus shown in FIG. 5 includes a selection valve 33 and a pump 34 for feeding the solution filled in the

reservoirs

8 and 9, a selection valve 35 used for feeding the separation column 1B, and a separation column 1C. A selection valve 36 used for liquid feeding, a switch valve 37 for controlling liquid feeding to the separation column 1C, and a switch valve 32 connected to the separation column 1B via a pipe on the opposite side to the switch valve 37 are provided. Except for this, the configuration is the same as that of the separation apparatus shown in FIG.

<Column design and control method for continuous processing>
For example, in the separation and purification of substances using an affinity column, (I) equilibration of the column, (II) adsorption of the target substance, (III) washing, (IV) elution of the target substance, (V) column It consists of a regeneration process. The sample solution is treated by (II) adsorption of the target substance. In other words, the other steps (I), (III), (IV), and (V) are not steps for processing the sample solution. In this separation apparatus, the sample solution can be processed continuously by shifting the processing steps in a plurality of packed columns as shown in FIG. Thereby, a large amount of sample solution can be processed efficiently in a short time, and the target substance can be separated and purified efficiently.
Moreover, in this separation apparatus, the same separation column can be repeatedly used up to the use frequency limit.

Furthermore, the separation column is divided into a plurality of columns at the column bed height (in the direction of the linear flow rate of the sample solution), so that the pressure loss applied to each column can be reduced and the pressure load applied to the column can be reduced. Here, the sum of the column bed heights of a plurality of columns is defined as the column bed height when the stationary phase having a capacity capable of processing the total amount of the mobile phase containing the substance to be separated is a single column. Thereby, a large amount of sample solution can be processed efficiently in a short time, and the target substance can be separated and purified efficiently.

When two or more split columns are used in this separation device, each separation column depends on the amount of sample solution to be processed and the time required for each step (I) to (V). It is preferable to do. A specific design method is described below.

1. How to determine the number of columns In order to continuously process the substances contained in the culture solution (above (I) to (V)), use two or more columns and shift the time ( II) It is necessary to carry out the adsorption process of the target substance (see Fig. 6). Here, when the number of columns is N, T1: column equilibration time, T2: target substance adsorption time, T3: washing time, T4: target substance elution time, T5: column regeneration time, Generally, by providing the number of columns so as to satisfy the formula (1), it is possible to continuously process the culture solution.

[Equation 1]
N × T2 ≧ (T1 + T2 + T3 + T4 + T5) Equation (1)

On the other hand, the time of each (I) to (V) can be determined as follows. However, the following method is an example and may be determined based on other criteria.

Method for Determining Column Equilibration Time T1 The column equilibration time can be determined as the time required to stabilize the baseline by monitoring the absorbance of the eluate during column equilibration. On the other hand, it may be the time required for the amount of liquid fed at the time of solvent replacement to reach 10 times the column volume.

Determination method of target substance adsorption time T2 If the linear velocity of liquid delivery to the column is determined, the time required for adsorption of the target substance can be determined by the following equation 2.
(Adsorption time) = (Column bed height) / (Linear velocity)

The linear velocity can be determined as follows. The column bed height and the linear velocity have the relationship shown in FIG. 7, and it is necessary to satisfy the condition of the range below the allowable pressure of the column (the curve P in the figure). Within this range, the antibody recovery rate is experimentally measured under various conditions of changing the column bed height and linear velocity, and determined by the column bed height and linear velocity that maintain a constant recovery rate (eg, 95%). can do. Instead of measuring the recovery rate, the dynamic binding capacity shown below may be measured.

An example of linear velocity measurement is shown below. An appropriate linear velocity was examined using a protein A column. After adding an IgG sample under various linear velocity conditions, washing with sodium phosphate (pH 7.0) containing 300 mM sodium chloride solution and 1.88 column volume (CV), then 20 mM sodium phosphate containing 300 mM sodium chloride solution (pH 2) .8), Stepwise elution was performed using 6.31 CV. The recovery rate was derived by quantifying the amount of IgG recovered at each linear velocity and taking the ratio with the amount of added IgG sample. In this example, the linear velocity at which the recovery rate was maintained at 90% or higher was determined as the appropriate linear velocity.

There are two types of carrier binding capacity: maximum binding capacity and dynamic binding capacity. The former represents the upper limit of the amount by which the carrier can collect the target molecule, and the latter is a numerical value representing how efficiently the carrier can be collected in the state where the sample to be purified is flowing. Since the carrier having a high dynamic binding capacity can recover a large number of target molecules at a high flow rate, the target molecule can be purified efficiently in a short time. On the other hand, for the maximum binding capacity, a standard protein solution is added to the column, and the absorbance of the eluate is monitored. The liquid feeding is continued until the absorbance of the eluate becomes the same as that of the added sample, and the amount of binding can be determined from the “eluted protein amount”. The maximum binding capacity is not affected by the addition flow rate.

An example of dynamic binding capacity measurement is shown below. The dynamic binding capacity was measured according to the following procedure. 10 mL of IgG diluted with PBS at a concentration of 10 mg / mL was sent with a syringe pump at a linear flow rate of 500 cm / h. The absorbance of the solution was measured from the outlet of the channel. The results are shown in FIG. In general, the dynamic binding capacity is often the amount of added protein at the time when the absorbance of the added sample or the leakage of 5% of the protein amount is observed, so 21.5 mg / mL is dynamic as shown in FIG. It can be seen that this is the binding capacity.

For reference, the method for obtaining the linear flow velocity (cm / h) is shown below.
Linear flow velocity (cm / h) = [flow velocity (ml / min)] x 60 / [column cross-sectional area (cm ² )] = [Z x 60 x 4] / [π x d ² ]
(Z = flow velocity, d = column inner diameter (cm))

Method for determining cleaning time T3 The effect of cleaning can be confirmed by measuring the pressure before and after cleaning. For example, before replacing the elution buffer with the washing solution or after washing with the washing solution, compare the pressure when the ultrapure water is replaced once and flow the ultrapure water, and the time until the pressure drops can be determined as the washing time. it can. As another method, the absorbance of the eluate is measured in the washing step, and the time until the baseline is stabilized can be determined as the washing time. Alternatively, it is possible to determine the time required for the flow of the cleaning liquid of a fixed multiple (for example, 10 times) of the column volume as the cleaning time.

¡Column washing needs to be considered in consideration of product integrity / safety and the lifetime of the purification carrier. For the purpose of column cleaning, the following cleaning agents can be used. However, it is not limited to these cleaning agents, and other cleaning agents can be used. That is, guanidine hydrochloride can be used as the cleaning agent. Guanidine hydrochloride is characterized by breaking hydrophobic interactions and dissolving precipitated and denatured proteins. An organic solvent (such as isopropanol) can be used as the cleaning agent. Organic solvents are characterized by the removal of lipids and hydrophobic impurities and the difficulty of removing precipitated raw materials. Furthermore, sodium hydroxide can be used as the cleaning agent. Sodium hydroxide solubilizes precipitated proteins, solubilizes lipids by alkaline hydrolysis, removes nucleic acids from the carrier, has a bactericidal action, and has the characteristics of degrading protein ligands. Among the above cleaning agents, sodium hydroxide is particularly preferable because it is effective for cleaning, disinfection, pyrogen inactivation, and virus inactivation.

Elution time of target substance T4
The time from when the antibody is adsorbed to the column and the eluate starts to flow until the absorbance due to antibody elution is reduced and returned to the baseline can be determined as the elution time.

Column regeneration time T5
As an example, 10 column volumes (CV) of 20 mM phosphate buffer, 0.5 M NaCl solution, and 50 mM EDTA solution (pH 7.4) are sent to remove metal ions, and then 15 CV ultrapure water is sent. The time required for this can be used as the column regeneration time. If the column is very dirty, it may be necessary to slowly feed 5CV of 1M NaOH solution and wash, and then add 15CV or more of neutral buffer to set the time required to return the column to neutrality. it can.

If ultrapure water is flowed after washing with an acid or alkali solution, acid or alkali may remain in the column and damage the support, so a washing step with NaCl can be performed after washing with acid or alkali. Damage to the carrier can be reduced.

2. Determination method of column volume Concentration of target substance in culture solution: Ct, volume of culture solution per batch: Vb, target substance dynamic adsorption amount per unit volume: Am, capacity per column: Vc, column Assuming that the number of N is N and the number of column reuse times is R, the relationship of Equation (2) is established.

[Equation 2]
Ct ・ Vb ≦ Am ・ Vc ・ R ・ N Formula (2)

By increasing the number of columns N, the equations (1) and (2) can be satisfied. However, as the number of columns N increases, the control system of the purification apparatus becomes more complicated. It is desirable that the number of columns N be the minimum. The stationary phase in the column is normally used repeatedly, but protein A in the stationary phase gradually deteriorates (desorbs or denatures) due to alkali treatment such as NaOH in the regeneration process, and the recovery amount of the target substance gradually increases. The number of reuse until the target recovery amount cannot be obtained is defined as the lifetime (the number of lifetimes). If the life varies depending on the stationary phase, the recovery amount can be monitored for each use, and the life until the specified recovery amount determined by the user cannot be obtained can be used. The number of reuses guaranteed by the manufacturer It is good also as lifetime. Since R is the number of column reuses (lifetime), the column capacity is (1 / R) of a normal column.

3. Column splitting The required column volume is the product of the capacity Vc per column and the number N of columns. A column having a volume of Vc · N is divided into N columns. A column with the same column bed height and a column cross-sectional area divided into 1 / N has a larger pressure loss than the column before the division. Therefore, it is desirable that the column bed is divided into at least two parts in the height direction. What is necessary is just to divide the column bed height and cross-sectional area so that the pressure loss is smaller than the upper limit of the design (see FIG. 9). FIG. 9A has the same cross-sectional area and is divided in the height direction. FIG. 9B shows the case where the height and the cross-sectional area are divided, and the divided columns match the specifications of the commercial column.

4). Column material (filler and column holder)
As described above, the separation column is packed with an amount of stationary phase that can process the entire amount of mobile phase to be processed. Thus, after using a separation column for a given batch, the separation column itself can be replaced and the next batch can be processed using a new separation column. In particular, as described above, when a plurality of separation columns are used, the pressure applied to the column can be reduced even if the mobile phase to be processed has a large capacity. Therefore, in this case, acrylamide, polypropylene, high-density polyethylene, polyethylene terephthalate, or the like that is used as a disposable material in other fields can be used as a material for the column holder. In addition, as a separation column, what used conventionally used glass and stainless steel as the material of a column holder can also be used.

Also, any conventionally used material can be used as the base of the stationary phase. For example, any of agarose and silica gel or a combination thereof can be used. In addition, the ligand that can specifically capture the substance to be separated is not particularly limited, and for example, Protein A, G, and Thiophilic interaction can be used.

<Application example for biopharmaceutical purification process>
FIG. 10 shows a system in which the separation apparatus according to the present invention is applied to a biopharmaceutical purification process. The system shown in FIG. 10 includes a medium receiving tank 101 filled with a culture solution in which cells that secrete and produce antibodies such as biopharmaceuticals are cultured, and an affinity column 102 having a stationary phase that specifically captures the antibody to be separated. And an eluent tank 106 filled with an eluent that elutes the antibody captured in the stationary phase. In the system shown in FIG. 10, downstream of the affinity column 102 are a fraction tank 109, a UF filtration device 105, a sterilization filter 108, a receiving tank 110, an anion exchange column 103, a fraction tank 109, a cation exchange column. 104 and the fraction tank 109 are installed in order. Further, in the system shown in FIG. 10, a cooling device 107 is disposed upstream of the affinity column 102.

First, a culture solution containing a biopharmaceutical such as an antibody is clarified by removing components such as cells by centrifugation or the like. Thereafter, a target biopharmaceutical such as an antibody is collected using the system shown in FIG. In this recovery step, the affinity column 102 continuously processes the culture solution according to the process chart shown in FIG. During this time, the packing material of the affinity column 102 can be reused according to the process chart shown in FIG. The stationary phase packed in the affinity column 102 has a capacity capable of processing the entire amount of the culture solution, and preferably the total amount of the culture solution is the upper limit of use. Here, the use upper limit amount is the maximum amount of mobile phase that can maintain the separation performance of the stationary phase.

Then, the culture sample solution that has undergone the recovery step is filled into a vial through intermediate purification and final purification. After one batch of the culture solution is completed, the affinity column 102 can be removed and incinerated as it is. About the next culture solution, the next preparation can be easily performed by connecting the new affinity column 102 to the line. Thus, by applying the present invention, it is not necessary to replace the packing material when the column is deteriorated, and the processing time can be shortened. In addition, by applying the present invention, a large number of culture solutions can be processed very easily without requiring a special apparatus or technique for packing a large amount of packing material into a large column.

DESCRIPTION OF SYMBOLS 1 ... Column, 2 ... Column, 3 ... Liquid feeding pump, 4 ... Liquid feeding pump, 5-9 ... Reservoir, 10-13 ... Switch valve, 14-15 ... Selection valve, 21-24 ... Switch valve, 31 ... Column, 32 ... Switch valve, 33 ... Selection valve, 34 ... Liquid feed pump, 35-36 ... Selection valve, 37 ... Switch valve, 38 ... Switch valve, 39 ... Computer, 40 ... Control device, 41-43 ... Switch valve, 44 to 46 ... UV detector, control device, 101 ... Medium receiving tank, 102 ... Affinity column, 103 ... Anion exchange column, 104 ... Cation exchange column, 105 ... UF filtration device, 106 ... Eluent tank 107 ... Cooling device 108 ... Disinfecting filter 109 ... Fraction tank 110 ... Receiving tank

Claims

A separation column having a stationary phase with a capacity capable of processing the entire amount of the mobile phase containing the substance to be separated, and the separation column is replaceable;
Separation apparatus characterized in that the number of times the stationary phase is used becomes the number of lifetimes in one batch of processing.
The separation column is composed of a plurality of columns including the stationary phase, and the total amount of the stationary phase packed in the plurality of columns is a capacity capable of processing the total amount of the mobile phase containing the substance to be separated. The separation device according to claim 1, wherein
A plurality of pipes connected to each of the plurality of columns, a plurality of switch valves respectively disposed on the plurality of pipes, and a control device for controlling communication of the pipes by the plurality of switch valves; The separation apparatus according to claim 2, further comprising:
3. The separation apparatus according to claim 2, wherein the total of the column bed heights of the plurality of columns is a column bed height when the stationary phase having the capacity is a single column.
The separation apparatus according to claim 1, wherein the capacity of the stationary phase is defined based on the total amount of substances to be separated contained in the mobile phase and the maximum adsorption capacity of the stationary phase.